Antigen

Antigen Class

TFs and others

Antigen

EBNA2

Cell type

Cell type Class

Blood

Cell type

GM12878

Tissue

blood

Lineage

mesoderm

Description

B-lymphocyte, lymphoblastoid, International HapMap Project - CEPH/Utah - European Caucasion, Epstein-Barr Virus

Sample

source_name

EBV transformed B cell line

biomaterial_provider

GM12878

cell line

GM12878

cell type

B cell

antibody

EBNA2 mouse monoclonal antibody (PE2) plus secondary rabbit anti-mouse

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Following cross-linking, glycine was added to a final concentration of 1.25 M and cells were washed in PBS and lysed in 300 ul of cell lysis buffer (85 mM KCl, 0.5% NP-40, 5 mM PIPES pH 8.0, 1 mM PMSF and EDTA-free protease inhibitor cocktail (Roche)) per 10 7 cells. Following a 10 minute incubation on ice, cell nuclei were pelleted by centrifugation at 8000 rpm for 5 minutes at 4ºC in a bench-top Microfuge. The nuclei were then resuspended in 200 µl of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8.0 plus protease inhibitors) per 10 7 cells and sonicated to reduce DNA length to between 200 and 600 bp. Each ChIP was carried out using 600 µl of chromatin from 30 x 10 6 cross-linked cells diluted 10-fold in IP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8.0, 167 mM NaCl) and pre-cleared with protein A sepharose beads. An input control sample was removed and EBNA 2 was precipitated using 48 µg EBNA 2-specific mouse monoclonal antibody (PE2) overnight at 4ºC with rotation followed by an additional 2 hr incubation with 60 µg rabbit anti-mouse antibodies (Dako). IgG control samples were incubated overnight with 48 µg of a 1:1 mix of sheep and mouse IgG (Santa Cruz Biotechnology). BSA-coated protein A sepharose beads were then added and immune complexes collected by rotation at 4ºC for 3 hrs. Beads were washed for 10 minutes at 4 ºC with rotation using a series of different wash buffers. Complexes were washed once in low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 150 mM NaCl), once in high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 500 mM NaCl), once in LiCl wash buffer (250 mM LiCl, 1% NP40, 1% Na deoxycholate, 1 mM EDTA, 10 mM Tris pH 8.0) and twice in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). Immune complexes were eluted in TE elution buffer (10 mM Tris pH 8.0, 5 mM EDTA, 1% SDS) at 65 ºC for 15 minutes. TE elution buffer was added to the input control sample and all samples were then incubated at 65 ºC overnight to reverse the crosslinks and samples were treated with 0.2 µg/ml RNAse A for 1 hr at 37 ºC to remove RNA. DNA was purified using QIAquick gel extraction and PB buffer (Qiagen). ChIP and input DNA (10 ng) was used to generate sequencing libraries with an NEBNext ChIP-seq library prep reagent set for Illumina using NEBNext Index primers. PCR-amplified samples (16-cycles) were separated on a 2% agarose gel in TAE buffer, visualized using SYBR-safe stain and a Dark Reader transilluminator (Clare Chemical Research). The region of the gel containing 150-350 bp DNA fragments was excised and DNA purified using a gel extraction kit (Qiagen). The library was quantified using an Agilent bioanalzer and subjected to 35bp single-end read sequencing with an Illumina Genome Analyzer IIx with 6 samples per lane.

Sequencing Platform

instrument_model

Illumina Genome Analyzer IIx

hg38

Number of total reads

19283397

Reads aligned (%)

33.5

Duplicates removed (%)

15.8

Number of peaks

1586 (qval < 1E-05)

hg19

Number of total reads

19283397

Reads aligned (%)

33.1

Duplicates removed (%)

16.2

Number of peaks

1731 (qval < 1E-05)